Project summary A major goal of genetics and evolutionary biology is to understand how changes in genotype affect phenotype. Genetic variants affecting fitness are especially informative for investigating evolution, since natural selection acts exclusively on these variants. By identifying specific variants that influence fitness, we can begin to understand the molecular mechanisms driving the incredible adaptations of all organisms to their respective environments. We recently developed an approach that allows us to edit genomes with unprecedented efficiency (~100%) and throughput. In our initial screen, we measured the fitness effects of 16,000 natural genetic variants differing between two strains of Saccharomyces cerevisiae. In this pilot experiment, we measured the effects of each variant in isolation, in a single condition. We found that nearly all strong fitness effects were from promoter variants, rather than protein-coding regions, and these were especially enriched at transcription factor binding sites. Here we propose to utilize this powerful system to investigate two types of interactions of fundamental importance: gene-by-environment (GxE) in Aim 1, and gene-by-gene (GxG, or epistasis) in Aim 2. Understanding the context-dependence of fitness effects will reveal key insights into the evolutionary process that would be unapproachable without our high-throughput precision genome editing technology. !